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(2023)ThesisDistinct localisation of macromolecular structures relative to cell shape is a common feature across the domains of life. One mechanism for achieving spatiotemporal intracellular organisation is the Turing reaction-diffusion system (e.g. Min system in the bacterium Escherichia coli controlling in cell division). In this thesis, I explore potential Turing systems in archaea and eukaryotes as well as the effects of subdiffusion. Recently, a MinD homologue, MinD4, in the archaeon Haloferax volcanii was found to form a dynamic spatiotemporal pattern that is distinct from E. coli in its localisation and function. I investigate all four archaeal Min paralogue systems in H. volcanii by identifying four putative MinD activator proteins based on their genomic location and show that they alter motility but do not control MinD4 patterning. Additionally, one of these proteins shows remarkably fast dynamic motion with speeds comparable to eukaryotic molecular motors, while its function appears to be to control motility via interaction with the archaellum. In metazoa, neurons are highly specialised cells whose functions rely on the proper segregation of proteins to the axonal and somatodendritic compartments. These compartments are bounded by a structure called the axon initial segment (AIS) which is precisely positioned in the proximal axonal region during early neuronal development. How neurons control these self-organised localisations is poorly understood. Using a top-down analysis of developing neurons in vitro, I show that the AIS lies at the nodal plane of the first non-homogeneous spatial harmonic of the neuron shape while a key axonal protein, Tau, is distributed with a concentration that matches the same harmonic. These results are consistent with an underlying Turing patterning system which remains to be identified. The complex intracellular environment often gives rise to the subdiffusive dynamics of molecules that may affect patterning. To simulate the subdiffusive transport of biopolymers, I develop a stochastic simulation algorithm based on the continuous time random walk framework, which is then applied to a model of a dimeric molecular motor. This provides insight into the effects of subdiffusion on motor dynamics, where subdiffusion reduces motor speed while increasing the stall force. Overall, this thesis makes progress towards understanding intracellular patterning systems in different organisms, across the domains of life.
(2023)ThesisThe Mediator complex plays a crucial role in transcription as a co-activator that regulates the expression of most RNA Polymerase II (Pol II) transcripts. Beyond its role in recruiting Pol II and the general transcription factors to form the Pre-initiation complex (PIC), Mediator has also been shown to impact the state of the chromatin and plays a role in the genomic organization. It also mediates functions such as DNA repair or RNA-splicing. It is still unknown how this protein complex manages multiple operations efficiently and can interact with an extremely high number of proteins inside the nucleus. How the Mediator complex establishes low affinity and high specificity protein-protein interactions is the focus of this study. The role of Mediator in transcription is vast; here, we focus on a specific interaction between Mediator and the transcription factor SOX18, whose role is crucial for cell fate in early embryogenesis. We unravelled how this TF interacts with the Tail module of Mediator and how this interaction is restricted to the transactivation domain of the protein. We used single-molecule techniques to reveal these protein-protein interactions via AlphaScreen proximity assay between SOX18 WT and synthetic constructs containing different regions of the protein: we also studied the interactions between Mediator and SOX18 pathological mutants. These mutations lead to the development of a disease called HLTRS, in which patients present anomalies in the development of their blood vessels and lymphatic system. It was crucial to understand how those mutations can affect the interactions with Mediator. We discover that mutations associated with truncations losing the TAD domain have an entirely different interaction pattern than those still conserving a portion. This study also touches on the role of Mediator and DNA repair via two specific subunits Med17 and Med20. Specifically, Med20 was described as presenting a series of mutants that leads to a disease presenting phenotypes comparable to Cockayne’s syndrome, a disease caused by DNA repair deficiencies. We study the PPIs within those mutants, and DNA repair proteins from four different DNA repair pathways (NER, NER-GG, NER-TCR and NEHJ); our results revealed that these mutations do not seem to affect the interactions with DNA repair proteins dramatically, but this appears to be sufficient to impair DNA repair. Thermostability assays against Med20 mutants and Med20 WT revealed similar behaviour within missense mutants, but early protein aggregation on the mutation led to a truncation. Finally, we investigated the role of virulent agents against Mediator: since Mediator is a multifaceted complex in charge of several essential functions on the cell, could viruses target them? We tested the ability of the two main proteases of SARS-CoV-2, Nsp3 and Nsp5, to cleave Mediator subunits in vitro. We discovered that out of 26 subunits tested, Nsp5 could cleave four (Med15, Med21, Med23 and Med28), and Nsp3 could cleave Med16, although no evidence was related Nsp3 to the nucleus as Nsp5. One of the cleaved proteins, Med23, acts as a coactivator of the signalling pathway WNT/β catenin and could significantly affect disease progression.
(2022)ThesisPrior to the emergence of cellular life, there must have been an abiotic analogue to the cell -- also known as the protocell. To flourish, they should have performed key functions enabling growth, and ensuring their survival and propagation. While researchers study a variety of model protocell systems, the study of model protocells assembled from fatty acid vesicles is lacking in comparison to the abundant literature on phospholipid vesicles. There is evidence that the existence of fatty acids predated life on Earth. Moreover, the highly dynamic nature of fatty acid vesicles is not well documented in the origins of life context. There is a need to explore the behaviour of fatty acid vesicles in systems which mimic real world environments. I explored the flocculation behaviour of vesicles in environments of mechanical stress, monovalent salt and particles including minerals. I also explored the effect of RNA-relevant divalent cations on fatty acid vesicles, as well as changes due to pH fluctuation. The findings of this project suggest that fatty acid vesicles are able to flocculate under quite low concentrations of monovalent salt, and that such flocs can incorporate particles including mineral particles which are relevant for catalysing RNA-related reactions. Furthermore, I found that after vesicles have been formed, they are surprisingly robust in conditions that inhibit vesicle formation. These results provide evidence that protocells assembled from simple building blocks such as fatty acids are quite robust and may persist in challenging environments, and that their flocculation behaviour may have co-localised other processes pertaining to the origins of life.
(2019)ThesisMulti-sensors integration is widely used to achieve different mapping and navigation objective. Among them, vision sensor could be recognized as one of the common sensors which contain large scene and feature information. Although this type of sensor has significant advantages in mapping and navigation, there still exist big challenges such as how to detect and remove mismatches among the image dense matching and improve the reliability of the integration result. This research aims to develop a quality control procedure to eliminate mismatches or outliers in the image dense matching procedure, and to evaluate the reliability and separability of these potential mismatches or outliers, which are measured with the Minimum Detectable Bias (MDB) and the Minimum Separable Bias (MSB), respectively. The experiments have shown that when the number of images is increased, the MDB and MSB will significantly decrease, which means the reliability and separability will be improved. The numerical results from some case studies are discussed in detail.
(2022)ThesisThere are studios all over the world where neurodiverse artists work together in a supportive way. This thesis is an inquiry into how art works in two of these organisations—Studio A in Sydney, Australia, and Project Art Works in Hastings, UK. It draws from traditions of narrative inquiry and ethnography to understand the lived experiences of the people at the heart of these studios, and the role that art has played in their storied lives. As such, the thesis contributes to knowledge in three ways: 1) It documents the important practices of makers and organisations whose work has not yet received significant critical or academic attention. It explores the dimensions of these practices that hold potential for reshaping normative understandings of both art and disability; 2) It conceptualises the role of art as a point of connection between neurodiverse people, and as a way of coming to express and understand lived experience. It maps the resonances across different fields that help articulate empathic encounters with and through art; 3) It demonstrates, through its written form, an ecological mode of creative inquiry that resists reductionism—an inquiry that is, like the practices it studies, embodied and relational. It interrogates the value, and ethical implications, of this mode of research. To contribute to knowledge in this way, the thesis assembles many forms of pre-existing knowledge, including the lived experience of its subjects, and the academic literature preceding it. It is grounded in an ecological understanding of cognition, informed by theories that help situate thought in the world, as a dynamic system of relationships between self, others, and the environment. It draws links between disability aesthetics, care ethics, and an ecological approach to empathy, through detailed insights into the social and aesthetic dynamics operating in the work of the two studios. These insights were built up over three years of fieldwork, including over one hundred interviews, and hundreds of hours spent looking, listening, and making alongside artists in the studio. This thesis is an invitation to enter the world of the studios, and of some of the people who work there. It offers a way of paying attention to art, and to other people, that is attuned to the senses, and that allows us to be comfortable with not knowing—or, knowing differently. It argues that this is a practice of ethical importance, in a world where both disability and art are still poorly understood.